By Herman S. Overkleeft, Bogdan I. Florea
This quantity makes a speciality of explorative activity-based proteomics,biomedical purposes of activity-based proteomics, and chemical options in activity-based proteomics delivering a concise review of activity-based protein profiling. Written within the hugely profitable Methods in Molecular Biology series structure, chapters comprise introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, with no trouble reproducible laboratory protocols, and pointers on troubleshooting and fending off identified pitfalls.
Authoritative and state-of-the-art, Activity-Based Proteomics: tools and Protocols goals to make sure profitable ends up in the extra learn of this very important field.
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Additional info for Activity-Based Proteomics: Methods and Protocols
16. 5. In the absence of a substrate, trypsin tends to cleave itself (autolysis). To prevent this, trypsin is stored in solutions at low pH and at low temperatures. 5 μg/μL solution at −20 °C until needed. 5. The reaction buffer must be able to buffer out 50 mM acetic acid or HCl. 17. Calculate beforehand how much of the working solution you will need and prepare only this amount plus a little pipetting surplus. For example, if you have 20 in-gel digests and you need 20 μL of the working solution per digest, prepare (20 + 2) × 20 μL = 440 μL of the working solution.
26. 27. 28. 29. 30. 31. 32. 45 Nat Chem Biol 5(9):616–624. 216 Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2(8):1896–1906. 261 White K, Bruckner JV, Guess WL (1973) Toxicological studies of 2-mercaptoethanol. J Pharm Sci 62(2):237–241 Zikolov P, Budevsky O (1973) Acid-base equilibria in ethylene glycol--I: definition of pH and determination of pk-values of acid-base indicators.
4. Add 20 μL of the 5 mM TBTA solution (final concentration in this case is ~100 μM) and vortex briefly. 5. Start the click reaction by adding 20 μL of the 50 mM CuSO4 solution (final concentration is around 1 mM) (see Note 24) and vortex briefly. Incubate the reaction in the dark (see Note 25) at room temperature for 1 h, while gently shaking. Occasionally, you may observe the formation of a light cloudy precipitate (see Note 26) which will, however, vanish upon addition of EDTA in the next step.